ES2640906T3 - Primate model of the family of cercopitecids infected by a strain of human genotype HBV - Google Patents

Abstract

Use of a strain of HBV isolated from a human D genotype comprising the sequence SEQ ID NO: 1 to infect a macaque of the Cercopithecinae subfamily to produce a primate model for human HBV disease.

Description

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DESCRIPTION

Primate model of the family of cercopitecids infected by a strain of human genotype HBV

[0001] The application describes a new non-human primate model of hepatitis B virus (HBV).

[0002] The five types of viral hepatitis, genotypes of hepatitis A, B, C, D, E, are now quite well known. In each case, the virus invades the liver and causes an inflammatory state with destruction of liver cells.

[0003] Hepatitis B is caused by a virus, the human hepatitis B virus (HBV). In most cases, HBV infection does not give rise to any symptoms and is responsible for asymptomatic acute hepatitis. Acute hepatitis is characterized by digestive disorders, abdominal pain, discolored urine, stool, abnormal stool, asthenia and jaundice. Acute hepatitis can develop into a fulminating form with rapid liver necrosis.

[0004] Viral infection can also become a chronic form, either in patients who have shown acute hepatitis, or in individuals for whom the infection was asymptomatic. 10% of infected adults and 90% of infected newborns become chronic carriers (Ganem, 1996; Maddrey, 2000). Chronic infections frequently progress to cirrhosis and liver cancer (Beasley, 1988). Chronic carriers have hepatic lesions of varying severity and an increased risk of developing cirrhosis and cancer of the primitive liver. In Asia and Africa, where infections are often chronic, primitive liver cancers represent a major public health problem. In addition, chronic carriers are virus reservoirs and allow them to spread, transposing the public health problem to a global problem.

[0005] The HBV virus was discovered by Blumberg et al, 1965. This virus carried in the blood can also be acquired through sexual contact or perinatal transmission. HBV is a small DNA virus with a diameter of 42 nm, which belongs to the group of hepatotropic DNA viruses (hepadnavirus) and is classified in the Hepadnaviridae family. Its genomic structure is remarkably compact. The virus comprises an outer envelope and a nucleocapside. The envelope consists mainly of three surface antigens (HBsAg: hepatitis B surface antigens) that play an important role in the diagnosis of HBV infections. The nucleocapside contains the nucleus antigen (HBcAg), a DNA polymerase / reverse transcriptase, as! as the viral genome, and the outer membrane carries the main antigenic determinant (epltopo) of the virus, the HBs antigen. The viral nucleus (approximately 28 nm in diameter) remains within the envelope that carries the main antigenic determinant (the "a" determinant).

[0006] HBV particles predominantly contain rcDNA with a complete negative chain and a partially synthesized positive chain. During the initiation of the infection, the virion's DNA is converted into a cccDNA that serves as a template for the transcription of an RNA intermediate, the pregenome. Next, a relaxed, partially double-stranded circular DNA genome of ~ 3-kb is synthesized by reverse transcription of the pregenome. The mechanism of DNA-directed DNA synthesis has been well characterized by genetic studies, as! as biochemicals, which have been described in several reviews. In contrast, the early events of the viral life cycle, including the entry, removal of the envelope, and the release of the viral genome in the cell nucleus, are not well understood. This is, in part, due to the absence until recently of cell lines that are susceptible to hepadnavirus infection.

[0007] Hepatitis B virus (HBV) genotypes have a characteristic geographical distribution. Hepatitis B virus (HBV) has been classified into 8 genotypes (AH) based on an intergroup divergence of 8% or more in the complete nucleotide sequence. Genotypes A and C predominate in the USA. However, genotypes B and D are also present in the USA. Genotype F predominates in South America and Alaska, while A, D and E predominate in Africa. Genotype D predominates in Russia and in all its previous domains, while in Asia, genotypes B and C predominate. By sequencing and phylogenetic analysis of local HBV isolates, the dominant HBV genotype in Tibet has been described It is a hybrid C / D (Chaoyin Cui et al., 2002).

[0008] Although an effective vaccine has been developed (see, for example, Lemon and Thomas, 1997, for a review), HBV infection remains a public health problem worldwide, with 400 million Chronic carriers of HBV making HBV infections the fourth leading cause of death from infectious diseases (Wright et al., 1993). Every year, almost 1 million people succumb to liver disease associated with HBV, especially cirrhosis and hepatocellular carcinoma. The number of chronic carriers already infected and the possible occurrence of vaccine escape mutants highlight the need for more effective treatments against HBV. In fact, existing treatments for chronic HBV infection are not yet satisfactory. Therefore, the development of better animal models to test new therapeutic approaches is highly desirable.

[0009] The lack of adequate in vitro infection systems and convenient animal models has greatly hindered the progress of HBV research.

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[0010] The generation of transfected HBV human hepatoma cell lines has contributed significantly to elucidate various aspects of viral replication and genetic expression. In addition, HBV has been successfully grown in primary cultures of human hepatocytes, but the susceptibility to infection is low and cultured hepatocytes become non-permissive for HBV very fast after seeding. Only recently, Gripon et al. (2002) describes a highly differentiated hepatoma cell line that, under specific conditions, appears to be susceptible to HBV infection.

[0011] Chimpanzees were the first animals that were found to be susceptible to HBV infection as demonstrated by the induction of acute infection and hepatitis in these animals after serum injection of hepatitis B virus carriers. human (Barker et al., 1973). They are the only primates known to develop a cellular immune response similar to that observed in humans acutely infected with HBV (Bertoni et al., 1998). However, they do not develop chronic liver disease. In addition, its use is strictly limited by its cost and by obvious ethical restrictions, its use is prohibited even in some countries, including France. In addition, they are endangered and unavailable (Will et al, 1982; Mac Donald et al., 2000). Natural HBV infection in chimpance, although it has been observed for a long time, has been demonstrated very recently (Hu et al, 2000).

[0012] The discovery of HBV-related viruses in the past, first in ducks, geese, herons, marmots, squirrels, and more recently in woolly monkeys, gibbons, gorillas and orangutans, offered opportunities for in vivo studies in several Animals with natural hepadnaviruses. However, most of the corresponding animals are difficult to handle in captivity or are not readily available or phylogenetically far from human beings. Newly developed humanized ducks, marmots, squirrels and mice are very good models for studying the viral cycle, but they are phylogenetically far from the human being with respect to the immune response, and do not develop hepatic inflammatory lesions, except for the marmot. Experimental and naturally occurring infections with HBV have been described in gibbons, orangutans and gorillas (Shouval, 1994; Warren et al., 1999; Lanford et al., 2000; Takahashi et al., 2000). However, there are no reports demonstrating the development of liver lesions and elevation of liver enzymes in these animals.

[0013] Based on the close phylogenetic relationship between tree mustards and primates, the species of tree musarana Tupaia belangeri has been analyzed for the study of HBV infection both in vitro and in vivo, taking advantage of these animals in the laboratory environment (Walter et al., 1996; Kock et al, 2001; von Weizsacker et al., 2004, Baumert et al, 2005). However, the inoculation of HBV in tree musaranas only causes a transient infection that results in a seroconversion and does not lead to a chronic carrier state. This is an important limitation for the use of this experimental animal model.

[0014] The groundhog is one of the most widely used animal models for HBV. This is due to the fact that HBV (groundhog hepatitis virus) is more similar to HBV in terms of genomic organization than avian hepadnaviruses and allows investigation of the entire viral life cycle in natural host hepatocytes, including during HCC development. VHM is morphologically indistinguishable from HBV and its genome shares approximately 60% nucleotide sequence identity with its human counterpart. However, a general disadvantage regarding the use of groundhogs is that they are genetically heterogeneous animals, difficult to breed in captivity and manipulate in many laboratories. Finally, the marmot immune system is not well characterized, and only very few monoclonal antibodies against marmot MHC molecules are available.

[0015] More recently, a new hepadnavirus with an intermediate host between humans and rodents has been isolated from a woolly monkey, Lagothrix lagotricha, a new world primate in danger of extinction. Phylogenetic analysis of the nucleotide sequences of the woolly monkey hepatitis B virus (WMVHB) genes indicated that the virus was distinct from HBV and probably represents a progenitor of human viruses. Unfortunately, the closest non-endangered relative of the woolly monkey, the black-handed spider monkey (Ateles geoffroyi), demonstrated that it was only marginally permissive to WMVHB, and only a minimal replication was observed after the inoculation of a chimpance with WMVHB

[0016] All currently available animal models have drawbacks: none are phylogenetically close to humans, they are not in danger of extinction, easy to use in the laboratory, and susceptible to developing hepatic lesions and innate or cellular immune response similar to that observed in humans acutely or chronically infected with HBV.

[0017] The development of a new experimental model closer to humans and susceptible to HBV infection is of particular importance, since it will represent an essential tool for new therapeutic strategy tests and study of HBV variants.

[0018] The HBV virus has always been considered highly specific to the host by the scientific community.

[0019] In 2002, Gheit et al. three M. sylvanus monkeys from Morocco have been intrahepatically inoculated with a human genotype D HBV DNA construct. They show that, after the direct transfection of DNA from the

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Human HBV of M. sylvanus liver, increasing amounts of circulating DNA are detected in the serum for several weeks with the presence of HBsAg, the presence of virus particles in the serum is confirmed and the increase in transaminase levels followed to the presence of virus markers in the serum. This demonstrates the appearance of HBV replication associated with acute hepatitis after intrahepatic transfection of M. sylvanus monkeys with HBV DNA.

The present inventors have surprisingly shown, for the first time, in two species of the family of cercopitecids, a natural infection by a strain of HBV of a human genotype.

[0021] Biological samples from a total of 50 cynomolgus macaques were included in the study (liver and serum). Subgenomic PCR and HBsAg detection allowed the identification of HBV markers among 42% (21/50) of the animals tested. Quantitative PCR indicated HBV viral loads among cynomolgus macaques ranging from 102 to 104 copies of HBV DNA per ml of serum. The complete HBV sequence circulating in these cynomolgus macaques showed a sequence near the human genotype D HBV sequence circulating among European intravenous drug users (U 95551 / gi 2182117).

[0022] In addition, in a sample of the liver of a Cercopithecus cephus that died in a French zoo of severe hepatitis, the presence of cover protelnas (AgHBs), capside protelnas (AgHBc) and DNA DNA sequences were also identified HBV

[0023] This provides a new approach to obtain primate models with HBV, closer to humans than currently available models, susceptible to HBV infection.

[0024] The invention in its broadest sense is defined as in the independent claims.

Use of a strain of human HBV to infect primate animal models

[0025] Therefore, the present application describes the use of a strain of HBV isolated from a human genotype or a strain of HBV hybrid of human genotypes to infect, preferably artificially infect, a non-human primate of the family of cercopitecides and to produce an animal model.

[0026] An "animal model" means herein an animal isolated from its natural environment that has been infected, preferably artificially infected, and can be used as a model for human HBV disease.

[0027] Preferably, said human HBV genotype is selected from the group consisting of human HBV genotype A, human HBV genotype B, human HBV genotype C, human HBV genotype D, human HBV genotype E, human F genotype of HBV, human G genotype of HBV and human H genotype of HBV.

[0028] More preferably, said human HBV genotype is selected from the group consisting of human HBV genotype A and human HBV genotype D.

[0029] Preferably, said hybrid HBV strain is a hybrid of two human genotypes as defined above.

[0030] In a preferred aspect, the HBV strain comprises or consists of a sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 1 .

[0031] The cercopitecidos are a group of primates, falling into the Cercopithecoidea superfamily in the Catarrhini clade. From the point of view of superficial appearance, they do not resemble apes in which the majority have tails (the name of the family means "ape with tail"), and unlike New World monkeys in that their tails are not prehensiles

[0032] The cercopitecides are native to Africa and Asia today, but they are also known from Europe in the fossil record. They include many of the most familiar species of nonhuman primates. The cercopitecidos include two subfamilies, the Cercopithecinae, which are mainly from Africa but includes the diverse genus of macaques that are from Asia and North Africa, and the Colobinae, which includes most of the genera of Asia, but also the colobus monkeys Africans

[0033] Preferably, the animal primate model isolated from the artificially infected family of cercopitecids is from the Cercopithecinae subfamily.

[0034] Preferably, the isolated primate animal model of the Cercopithecinae subfamily is a macaque, more preferably of the species Macaca sylvanus or Macaca cynomolgus, or a guenon, more preferably of the species Cercopithecus cephus.

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[0035] In a specific aspect, baboons are excluded from the scope of the cercopitecides described herein.

[0036] "Isolated" when used in reference to HBV strains and / or nucleotide sequences described herein means that the strain or nucleotide sequence has been subjected to at least one stage of body fluid purification and / or natural tissue or that is not present in its native environment. Alternatively, the strains may be maintained in body fluid and / or isolated tissue or may be in the form of nucleotides. Typically, this means that the virus strain or nucleotide sequence is free of at least one of the host proteins and / or host nucleic acids. In general, the virus strain or sequence of isolated nucleotides is present in an in vitro environment. "Isolated" does not mean that the virus strain or nucleotide sequence must be purified or homogeneous, although such preparations fall within the scope of the term. "Isolated" simply means elevated to a degree of purity, to the extent required with exclusion of product of nature and accidental anticipations of the scope of the claims. "Isolated" means that it includes any biological material taken either directly from an infected human or animal, or after cultivation (enrichment).

[0037] A "strain" means a genetic variant or subtype of a virus.

[0038] A "HBV genotype" is a HBV group based on 92% homology or more in the complete genomic nucleotide sequence.

[0039] The degree of "homologla" is established by registering all the positions for which the nucleotides of the two sequences compared are identical, in relation to the total number of positions.

[0040] A "hybrid strain a / p" means a strain whose genomic DNA sequence results from a recombination between a genomic nucleotide sequence of genotype a and a genomic nucleotide sequence of genotype p.

[0041] The term "recombination" means in this document the mechanism by which genes located in different homologous genomic sequences are found in the same recombinant genomic structure.

[0042] A "genomic nucleotide sequence of human HBV genotype A" means the total genetic material of a HBV strain of human genotype A.

[0043] "Human HBV genotype l" means a strain of HBV classified in the human genotype l.

[0044] "Artificially" means that the encounter between the virus and the animal does not occur randomly, but is a forced encounter (that is, the animal is inoculated with the HBV strain or HBV nucleotide sequence by human intervention ).

[0045] "Infected" means in this document the development of signs of infection.

[0046] "Signs of infection" include the presence of HBV genomic DNA in the liver or serum, the presence of viral proteins in the liver or serum and / or a viral load of at least 102 genomic copies / ml

[0047] The presence of HBV genomic DNA can be assayed by nucleic acid or serum point transfer or by PCR followed by Southern blotting followed by hybridization with a specific HBV probe, or by Northern blot analysis or by RT PCR. for RNA analysis. The presence of viral proteins can be tested by immunofluorescence, FACS or Western blotting assays, immunoassays (ELISA).

[0048] The viral load can be measured by real-time PCR.

[0049] Preferably, the viral load of HBV in the serum of an animal model according to the invention is between 102 and 108 copies / ml, more preferably between 103 and 108 copies / ml, most preferably between 104 and 108 copies / ml

Use of a primate from the family of cercopitecids to make an animal model infected with HBV

[0050] A further object described herein is the use of an isolated primate from the family of cercopitecids to make an animal model infected by a HBV strain of a human genotype or a hybrid HBV strain of two human genotypes.

[0051] The application also describes the use of a primate isolated from the family of infected cercopitecides, for example artificially infected, by a HBV strain of a human genotype or a hybrid HBV strain of two human genotypes as an animal model for the infection for human HBV disease.

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[0052] Preferably, said human HBV genotype is selected from the group consisting of human HBV genotype A, human HBV genotype B, human HBV genotype C, human HBV genotype D, human HBV genotype E, human F genotype of HBV, human G genotype of HBV and human H genotype of HBV.

[0053] More preferably, said human HBV genotype is selected from the group consisting of human HBV genotype A and human HBV genotype D.

[0054] Preferably, said strain of HBV hybrid is a hybrid of two human genotypes.

[0055] In a preferred aspect, the HBV strain comprises or consists of a sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 1 .

[0056] Preferably, the primate isolated from the cercopitecid family is from the Cercopithecinae subfamily.

[0057] Preferably, the isolated primate animal model of the Cercopithecinae subfamily is a macaque, more preferably of the species Macaca sylvanus or Macaca cynomolgus, or a guenon, more preferably of the species Cercopithecus cephus.

[0058] In a specific aspect, baboons are excluded from the scope of the cercopitecides described herein.

[0059] An "isolated primate" means a primate isolated from its natural environment.

HBV models of nonhuman primates

[0060] A further object described herein is an animal primate model of the family of cercopitecids infected with a strain of HBV of a human genotype or a strain of HBV hybrid of human genotypes.

[0061] Preferably, said primate animal model of the cercopitecer family is artificially infected with a HBV strain isolated from a human genotype or a hybrid HBV strain from human genotypes.

[0062] Preferably, said human HBV genotype is selected from the group consisting of human HBV genotype A, human HBV genotype B, human HBV genotype C, human HBV genotype D, human HBV genotype E, human F genotype of HBV, human G genotype of HBV and human H genotype of HBV.

[0063] More preferably, said human HBV genotype is selected from the group consisting of human HBV genotype A and human HBV genotype D.

[0064] Preferably, said strain of HBV hybrid is a hybrid of two human genotypes.

[0065] In a preferred aspect, the HBV strain comprises or consists of a sequence of at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 1 .

[0066] Preferably, the primate isolated from the cercopitecid family is from the Cercopithecinae subfamily.

[0067] Preferably, the isolated primate animal model of the Cercopithecinae subfamily is a macaque, more preferably of the species Macaca sylvanus or Macaca cynomolgus, or a guenon, more preferably of the species Cercopithecus cephus.

[0068] In a specific aspect, baboons are excluded from the scope of the cercopitecides described herein.

[0069] Preferably, the animal primate model has a viral load of HBV in the serum between 102 and 108 copies / ml, more preferably between 103 and 108 copies / ml, most preferably between 104 and 108 copies / ml.

[0070] An additional object described in this document is a method of providing a primate animal model, comprising the following steps consisting of:

- isolate an animal from the family of cercopitecids;

- infecting said animal with a strain of HBV isolated from a human genotype or a strain of HBV hybrid from human genotypes;

- test a viral load of at least 102 genomic copies of HBV / ml of serum.

Nucleotide Sequences and Expression Procedures

[0071] Another object described herein is a nucleotide sequence or a nucleic acid that comprises or consists of a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ iD NO: 1, or one

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complementary sequence of it.

[0072] SEQ ID NO: 1 is the genomic nucleotide sequence of the HBV strain isolated by the inventors in Macaca cynomolgus of Mauritius. This sequence has been shown to be classified in the human D genotype, that is, that it has more than 92% homologla with the genomic sequence of a human D genotype.

[0073] The terms "polynucleotide", "polynucleic acid", "nucleic acid", "nucleic acid sequence", "nucleotide sequence", "nucleic acid molecule", "oligonucleotide", "probe" or "primer" , when used herein, refer to nucleotides, ribonucleotides, deoxyribonucleotides, nucleotides of blocked peptides or nucleotides, or a combination thereof, in a polymeric form of any length or any form (eg, branched DNA). Such terms also include double chain (ds) and single chain (ss) polynucleotides, as! as triple chain polynucleotides. Such terms also include modifications of known nucleotides, such as methylation, cyclization and "caps" and substitution of one or more of the natural nucleotides by an analog, such as inosine or by non-amplifiable monomers, such as HEG (hexethylene glycol).

[0074] Ribonucleotides are indicated as PNT, deoxyribonucleotides as dNTP, and dideoxyribonucleotides as ddNTP.

[0075] Nucleotides may generally be radioactively labeled, chemiluminescent, fluorescent, phosphorescent, or with infrared dyes or with a Raman tag with a larger surface or plasmonic resonant particle (PRP).

[0076] By a nucleotide having a sequence of at least, for example, 95% "identifies" a query sequence, it is understood that the sequence of the object nucleotide is identical to the query sequence except that the sequence of object nucleotide may include up to five nucleic acid alterations per 100 nucleic acids of the query sequence. In other words, to obtain a nucleotide having a sequence at least 95% identical to a query, up to 5% (5 of 100) of the nucleic acids in the subject sequence may be inserted, removed or substituted by another nucleic acid.

[0077] The procedures for comparing the identity and homology of two or more sequences are well known in the art. You can use, for example, the "needle" program, which uses the global alignment algorithm of Needleman-Wunsch (Needleman and Wunsch, 1970 J. Mol Biol. 48: 443-453) to find the optimal alignment (including gaps ) of two sequences when considering its entire length. The needle program is, for example, available on the website ebi.ac.uk. The percentage of identity described herein is preferably calculated using the EMBOSS :: needle (global) program with a "Gap Open" parameter equal to 10.0, a "Gap Extend" parameter equal to 0.5, and a Blosum62 matrix

[0078] A further object described herein refers to a vector comprising a nucleotide sequence described herein placed under the control of the elements that ensure the expression of the nucleotide sequence.

[0079] "Elements that ensure the expression of said nucleotide sequence" refers in particular to the elements necessary to ensure the expression of said nucleotide sequence after its transfer to a target cell. It applies in particular to promoter sequences and / or regulatory sequences that are effective in said cell, and optionally the required sequences that allow a polypeptide to be expressed on the surface of the target cells. The promoter used can be a viral, ubiquitous or tissue specific promoter, or a synthetic promoter. As examples we can mention the promoters, such as the promoters of the RSV virus (Rous sarcoma virus), MPSV, SV40 (Ape virus), CMV (Cytomegalovirus) or vaccinia virus. In addition, it is possible to select a specific promoter sequence for a given cell type, or that can be activated under defined conditions. The literature contains a large volume of information related to these promoter sequences.

[0080] The terms "vector", "donation vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (eg, a foreign gene) can be introduced into a host cell, in order to transform the host and promote the expression (for example, transcription and translation) of the sequence introduced. Vectors include plasmids, phages, viruses, etc .; They are discussed in greater detail below.

[0081] The vectors typically comprise the DNA of a transmissible agent, into which the foreign DNA is inserted. A common way to insert a segment of DNA into another segment of DNA involves the use of enzymes called restriction enzymes that cleave DNA at specific sites (specific nucleotide groups) called restriction sites. A "cassette" refers to a DNA coding sequence or segment of DNA that encodes an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure that the cassette insertion in the appropriate reading frame. Generally, the foreign DNA is inserted into one or more restriction sites of the vector DNA, and then transported by the vector into a host cell along with the transmissible vector DNA. A segment or sequence of DNA that has DNA inserted or added, such as an expression vector, can also be called a "DNA construct." A common type of vector is a "plasmid," which is generally a self-contained molecule.

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of double-stranded DNA, usually of bacterial origin, that can easily accept additional (foreign) DNA and that can easily be introduced into a suitable host cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for insertion of foreign DNA. The coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme. The promoter DNA is a DNA sequence that initiates, regulates, or in any case mediates or controls the expression of the coding DNA. The promoter DNA and the coding DNA can be from the same or different genes, and can be from the same or from different organisms. A large number of vectors, including plasmids and fungal vectors, have been described for replication and / or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clontech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wl), pRSET or pREP plasmids (Invitrogen, San Diego, CA), or pMAL plasmids (New England Biolabs, Beverly, MA), and many appropriate host cells, using procedures described or cited herein or otherwise known to those of skill in the relevant art. Recombinant donation vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, for example, antibiotic resistance, and one or more expression cassettes. Expression vectors may comprise viral nucleic acid that includes, but is not limited to, vaccinia virus, adenovirus, retrovirus and / or adeno-associated virus nucleic acid. The nucleotide sequence or vector described herein can also be in a liposome or a delivery vehicle that can be collected by a cell through receptor-mediated endocytosis or other type of endocytosis.

[0082] The terms "express" and "expression" means allowing or causing information in a gene or aDn sequence to manifest itself, for example by producing a protein by activating the cellular functions involved in the transcription and translation of a gene. or corresponding DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product", such as a protein. The expression product itself, for example the resulting protein, can also be said to be "expressed" by the cell. An expression product can be characterized as intracellular, extracellular or secreted. The term "intracellular" means something that is inside a cell. The term "extracellular" means something that is outside a cell. A substance is "secreted" by a cell if it appears to a significant extent outside the cell, from somewhere inside or outside the cell.

[0083] Another object described in this document is a host cell that contains a vector described in this document, for example, by transfection, transformation or infection.

[0084] This host cell originates from a prokaryotic or eukaryotic organism.

[0085] Numerous tools have been developed for the introduction of several heterologous genes and / or vectors into cells, especially mammalian cells. These techniques can be divided into two categories: the first category involves physical techniques, such as microinjection, electroporation or particle bombardment. The second category is based on the use of techniques in molecular and cellular biology through which the gene is transferred with a biological or synthetic vector that facilitates the introduction of the material into the cell in vivo. Currently, the most efficient vectors are viral vectors, especially adenoviral and retroviral vectors. These viruses have natural properties to cross the plasma membranes, prevent the degradation of their genetic material and introduce their genome into the cell nucleus. These viruses have been widely studied and some are already being used experimentally in human applications in vaccination, immunotherapy, or to compensate for genetic deficiencies. However, this viral approach has limitations, due in particular to the restricted capacity for cloning in these viral genomes, the risk of propagation of viral particles produced in the organism and the environment, the risk of artifact mutagenesis through insertion. in the host cell in the case of retroviruses, and the possibility of inducing a strong inflammatory immune response in vivo during treatment, which limits the possible number of injections (McCoy et al, 1995, Human gene Therapy 6: 1553-1560 ; Yang et al, 1996, Immunity 1: 433-422). There are alternatives to these viral vector systems. The use of non-viral procedures, for example co-precipitation with calcium phosphate, the use of receptors that mimic the viral systems (for a summary, see Cotten and Wagner 1993, Current Opinion in Biotechnology, 4: 705-710), or the use of polymers, such as polyamidoamines (Haensler and Szoka 1993, Bioconjugate Chem., 4: 372-379). Other non-viral techniques are based on the use of liposomes, whose efficacy for the introduction of biological macromolecules, such as DNA, RNA, proteins or pharmaceutically active substances, has been widely described in the scientific literature. In this area, the teams have proposed the use of cationic lipids that have a strong affinity for cell membranes and / or nucleic acids. In fact, it has been shown that a nucleic acid molecule itself was capable of crossing the plasma membrane of certain cells in vivo (WO 90/11092), the effectiveness in particular depending on the polyanionic nature of the nucleic acid. Since 1989 (Felgner et al, Nature 337: 387-388) cationic lipids have been proposed to facilitate the introduction of large anionic molecules, which neutralize the negative charges of these molecules and favor their introduction into the cells. Several teams have developed cationic liquids of this type: DOTMA (Felgner et al, 1987, PNAS 84: 7413-7417), DOGS or Transfectam (TM) (Behr et al, 1989, PNAS 86: 6982-6986), DMRIE and DORIE (Felgner et al, 1993 methods 5: 67-75) DC-CHOL (Gao and Huang 1991, BBRC 179: 280-285), DOtAp (TM) (McLachlan et al, 1995, Gene Therapy 2: 674-622) or Lipofectamine T, and the other molecules described in patents WO9116024, WO9514651, WO9405624. Other groups have developed cationic polymers that facilitate the transfer of macromolecules, especially anionic macromolecules, into the cells. WO 95/24221 describes the use of dendritic polymers, WO96 / 02655 describes

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the use of polyethyleneimine or polypropyleneimine and United States patent documents. No. 5595897 and FR2719316 describe the use of polylysine conjugates.

[0086] The term "host cell" means any cell of any organism that is selected, modified, transformed, produced, or used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell. of a gene, a DNA or RNA sequence, a protein or an enzyme. Guest cells may also be used for screening or other assays, as described below.

[0087] The term "expression system" means a compatible host cell and vector under suitable conditions, for example, for the expression of a protein encoded by foreign DNA carried by the vector and introduced into the host cell. Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Suitable cells include hepatocyte cells. Most preferably, a host cell described herein is a HuH7, HepG2 or HepaRG hepatoma cell line, human or non-human primate hepatocytes in primary culture or any hepatocyte cell line.

Procedures for evaluating a therapeutic agent or vaccine

[0088] Another object described in this document is a procedure for evaluating a therapeutic agent according to which an animal model described herein is administered with defined doses, in a dose or in repeated doses and at specific time intervals, of a therapeutic agent. or a process or a diagnostic agent or process, biological samples are taken before and at defined time intervals after the administration of said therapeutic agent and qualitative and quantitative measurements of the HBV viral proteins in said samples are carried out and compared. Biological

[0089] The therapeutic agents or processes can affect any stage of the infection: membrane fixation, phagocytosis, decapsidation, DNA replication, viral protein maturation, virion assembly. They include nucleoside analogs, viral protease inhibitors, glycosylation inhibitors, antisense oligonucleotides, ribozymes, and immonomodulatory compounds. Diagnostic agents or processes include assays for the detection of the presence or amounts of lymphokines, cytokines, chemokines, antigens, antigen epltopos, particular antibodies or other biological macromolecules associated with HBV infection.

[0090] A further object described herein refers to a procedure for evaluating a vaccine according to which a primate animal of the cercopitecer family is administered with defined doses, in a dose or in repeated doses and at specific intervals of At the time of a vaccine, said animal is artificially infected with a strain of HBV isolated from a human genotype or a strain of HBV hybrid from two human genotypes, biological samples are taken before and at defined intervals of time after infection and carried out and qualitative and quantitative measurements of HBV viral proteins in these biological samples are compared.

[0091] Vaccines include live attenuated HBV, inactivated HBV and vaccine compositions comprising subunit proteins, especially recombinant proteins, DNA encoding viral genes, alone or in combination with immunomodulatory compounds.

[0092] As intended herein, the term "therapeutic" means the ability of a substance to treat a pathological reaction to HBV infection.

[0093] As intended herein, the term "vaccine" refers to the ability of a substance to prevent a pathological reaction to HBV infection.

[0094] In the context of the invention, the terms "treat," "treating," or "treatment," mean reversing, alleviating, or inhibiting the course of a pathological reaction or one or more symptoms thereof.

[0095] In the context of the invention, the terms "prevent" or "prevention" means the onset of a pathological reaction or one or more symptoms thereof.

[0096] The evaluation of the efficacy of a therapeutic agent and the ex vivo therapeutic monitoring are determined as follows: the therapeutic agents to be tested for therapeutic activity and / or for therapeutic monitoring are administered by various routes, such as intramuscular , subcutaneous or other vlas. Several doses are administered. One or more administrations can be carried out with different time intervals between each administration ranging from a few days to a few years. Biological samples are taken at defined time intervals after administration of the therapeutic agent, preferably blood and serum. Various analyzes are carried out on these samples. Just before the first administration of the therapeutic agent, said samples are taken and the same analyzes are also performed. The classical clinical examination and biological examination are also carried out in parallel with the complementary analyzes described below, at different analysis times.

[0097] The following analyzes are carried out: the qualitative and quantitative measurement of HBV viral proteins

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in the serum or in the blood by ELISA and / or Western blotting, using antibodies or antibody fragments that are capable of binding to at least one of the proteins or one of its fragments and / or measuring the activity of said proteins and / or or assay of antibodies specific for the proteins of interest or their fragments in the blood or serum samples by ELlSA and / or Western blotting using an isolated and purified natural protein or a fragment of the natural protein and / or a synthetic protein or a fragment of said synthetic protein or a synthetic polypeptide, and / or assay of the cellular immune response induced against the protein or proteins of interest and any immunogenic peptide derived from these proteins, as described above, and / or the detection of DNA fragments and / or RNA encoding the protein or proteins of interest or a fragment of said proteins of interest by nucleotide hybridization by techniques that are familiar to u A person skilled in the art (Southern transfer, Northern transfer, ELOSA "Enzyme-Linked Oligosorbent Assay" (Katz JB et al., Am. J. Vet. Res., 1993 Dec; 54 (12): 2021-6 and Franpois Mallet et al, Journal of Clinical Microbiology, June 1993, p. 14441449)) and / or by DNA and / or RNA amplification, for example by PCR, RT-PCR, using nucleic acid fragments encoding the protein or proteins of interest, and / or by tissue biopsy, preferably of the liver, and the observation of markers of HBV infection. It can be performed, for example, by immunohistochemistry or immunofluorescence for the detection of HBVAg in the liver, or by in situ hybridization or by PCR for the detection of HBV DNA in the liver sections.

[0098] The invention will be further illustrated in view of the following figures and examples.

FIGURES

[0099]

Figure 1: VHB transcription and translation map. The figure shows the physical map of the HBV genome. The inner circle represents rcDNA with the reverse transcriptase attached to the 5 'end of the complete negative strand DNA (solid sphere) and a blocked RNA oligomer attached to the 5' end of the incomplete positive strand DNA (solid half sphere). The positions of the direct repetitions, DR1 and DR2, as! as the positions of the two enhancers, EN1 and EN2, are indicated. The outer circle represents the three main viral RNAs, the nucleus (C) or pgRNA, the pre-S (L) mRNA, and the mRNA. The 3 'common ends of the three mRNAs are indicated by the letters A. No the possible mRNA X that covers the coding region of X is shown in the figure and ends at the site indicated for the other three mRNAs. The four codification regions of proteins are shown between the internal and external calculations. They include the prenucleus (PC) and nucleus genes, the polymerase gene, and the X gene. The envelope genes pre-S1 (L), pre-S2 (M), and the surface (S) overlap with the polymerase open reading frame.

Figure 2: Cynomolgus macaque HBV isolation strategy.

Figure 3: HBV genome agarose gel electrophoresis amplified by PCR (Gunther et al, 1995) and Southern blot hybridization.

Figure 4: HBV gene X sequence of macaque cynomolgus in the phylogenetic tree of HBV X gene in humans and nonhuman primates.

Figure 5: IHC labeling of HBsAg (A) or IF labeling of HBsAg in the sections of C. cephus (B) and M. cynomolgus (C) with anti-HBs antibodies (Dako, F) (Amplification: X20). Yellow arrows indicate marked hepatocytes.

Figure 6: Southern analysis of HBV PCR products obtained from sucrose gradient fractions of 22.5%, 42.5% and 48%.

Figure 7: Observation by electron microscopy of "viral" particles in the 42.5% sucrose gradient fraction positive for HBV DNA and PCR.

Figure 8: Follow-up of infection markers in sylvanus macaques BL12, BL13, BL14.

Figure 9: Southern blot analysis of PCR products (145 bp and 118 bp) in HBV S and C genes. Figure 10: IF labeling of HBcAg in liver sections of M. sylvanus (BL14). The arrows show HBcAg positive cells (X20).

Figure 11: Southern blot analysis of HBV PCR products in the S gene (118 bp). Follow-up from day 1 to 10 after infection.

Figure 12: detection of HBsAg in hepatocytes infected with supernatant in primary culture of M cynomolgus. Test cut-off value was 1.

Figure 13: Follow-up of AgHB infection markers and transaminases in sylvanus macaques BL01, BL3, BL04 after inoculation with a set of highly viremic human sera (109 copies / ml).

Figure 14: Follow-up of HBV and transaminase infection markers in sylvanus BL12, BL13, BL14 macaques after inoculation with a set of Cynomolgus macaque sera found naturally infected with HBV (103 copies / ml).

Examples

MATERIAL AND PROCEDURES Animals

[0100] Macaques. Serum and liver samples from 50 cynomolgus macaques come from a large crla facility in Mauritius. Serum and liver samples were obtained from twenty M wild-caught sylvanus (Middle Atlas Mountains), quarantined and kept at the Pasteur Institute in Casablanca

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(Morocco). All animals were negative for serological markers of infection with hepatitis A, C, HTLV I and HTLV II viruses.

[0101] Cercopithecus cephus. A sample of liver from a C. cephus who died in a French zoo of severe hepatitis was frozen at necropsy and kept at -80 ° C for further study. No other organ, except the liver, showed no injury.

Serology

[0102] MONLISA AgHBs Plus (BIORAD, Marne La Coquette, France) was used for the detection of HBsAg. VIDAS II HBs Ag Ultra was also used to detect HBs, as! such as ORTHO HBsAg Elisa Test system 3 (Ortho-Clinical Diagnostics, Levallois Perret, F), VIDAS HBCT II for anti-HBc, VIDAS HBST for anti-HBs, VIDAS HBeAg for the detection of HBeAg (bioMerieux, Lyon, F).

HBV DNA detection by polymerase chain reaction and Southern blot hybridization

[0103] Nucleic acids of 140 ml of serum or 10 mg of liver tissue were extracted with QiaAmp extraction kit respectively (Qiagen, Courtaboeuf, F) and pure master RNA and complete RNA purification kit (Epicenter, Le Perray, F ). Extraction with phenol chloroform was also performed by a procedure described in detail elsewhere (Jilbert et al., 1992).

[0104] Primers for PCR amplification were selected from sequences that overlap the nucleus and the surface gene, which are highly conserved among all human HBV genotypes (Gheit et al., 2002). A region of 118 bp in the surface gene was amplified (sense: 5'-GGA GTG GGC CTC AGC CCG TTT CTC-3 reverse: 5'-GCC CCC AAT ACC ACA TCA TCC ATA-3 '). A region of 145 bp in the nucleus gene was also amplified (sense: 5'-TCG GAG TGT GGA TTC GCA CTC CTC-3 '; reverse: 5' GAT-TGA GAC CTT CCT CCT CTG CGA GGA-3 ').

[0105] The specificity of the amplified bands was confirmed by Southern blot hybridization using a random barley HBV DNA probe labeled with 32P. Hybridization conditions were 50% formamide / 7% SDS / 0.25 M sodium phosphate, pH 7.2 / 0.25 M NaCl / 1 mM EDTA at 42 ° C 20 minutes twice, followed by washing in 2X-0.5X SSC-0.1% SDS at 65 ° C 10 minutes.

HBV DNA isolation and viral load quantification by real-time PCR

[0106] 100 ml VHB DNA of supernatant was extracted using the high purity viral nucleic acid kit according to the manufacturer's instructions (Roche, Grenoble, France). The quantitative analysis of the viral load was performed using real-time PCR (LightCycler, Roche, Grenoble, France). The primers were designed with Oligo5 software (MedProbe, Oslo, Norway) to target nucleic acid sequences within the C gene that are conserved among all known HBV (A-G) genotypes.

[0107] To quantify HBV DNA in the serum of HCV patients with hidden HBV, a real-time PCR assay (Roche Diagnostic Corporation, Mannheim, Germany) was performed using primers (CASB and CSB) that recognize highly conserved sites in the HBV C gene region. The sensitivity threshold of this assay (10 viral copies / ml) was evaluated according to the standards and expressed in genomic copies / ml. A PCR reaction was carried out using a total volume of 20 ml including 10 ml of DNA template, 2 ml of Light Cycler DNA master hybridization mixture (Taq DNA polymerase, reaction buffer, dNTP mixture and 10 mM of MgCh), 3.2 ml of 25 mM MgCl2, 0.3 ml each of the 20 mM primers. The samples were loaded into disposable capillaries, centrifuged, and placed in the Light Cycler with the following program:

1) DNA denaturation and activation of FastStart polymerase: 95 ° C for 10 min. slope 20 ° C / s. 2) 45 cycles of denaturation at 95 ° C for 15 seconds, hybridization at 58 ° C for 5 seconds, and extension at 72 ° C for 15 seconds. The programmed temperature transition speed was 20 ° C / second. Real-time PCR monitoring was achieved by measuring the fluorescence at the end of each cycle. 3) Fusion curve: a single fusion cycle was generated by maintaining the reaction at 95 ° C for 0 seconds, then at 65 ° C for 15 seconds, followed by slow heating at a transition rate of 0.1 ° C / s to 95 ° C. 4) Refrigeration: 40 ° C for 30 seconds with a slope of 20 ° C / second.

[0108] Fluorescence monitoring occurred at regular intervals during the hybridization phase and continuously throughout the fusion phase.

[0109] For each development, a standard curve was generated in a 5-log range (from 10 to 50,000 copies / ml) by serial dilutions of the biological standard of HBV DNA in the Versant HBV DNA kit (Bayer diagnostics , Puteaux, F). The fusion curve and quantitative analysis were carried out by using Light Cycler 3.5 analysis software following the manufacturer's instructions (Roche Diagnostics, Meylan, France).

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Amplification by rolling circle

[0110] The circular DNA template was denatured to become a single chain. After the exonuclease protected primers bind to DNA at multiple sites, they are extended by Phi29 DNA polymerase. Next, the displacement activity of the Phi29 DNA polymerase chain causes the nascent chain to shift when Phi29 reaches an extended primer in the 3 'direction. Continued elongation and chain displacement result in branching and exposure of new binding sites for primers. A cascade of initiation events results in exponential amplification and generates copies of the double-stranded template DNA, of high molecular weight, repeated in tandem.

[0111] The appropriate amounts of DNA were mixed with 9 phosphorothioate modified primers (Table 1) at a concentration of 10 pM each and 1X Phi29 buffer (New England Biolabs, St Quentin in Yvelines, F) in a final volume of 10 pl. The DNA mixture was denatured at 95 ° C for 3 minutes. Then, it was cooled to room temperature by doing some intermediate steps, namely: 50 ° C for 15 seconds, 30 ° C for 15 seconds and 20 ° C for 10 minutes. Finally, the denatured product was placed on ice before proceeding with the RCA reaction.

[0112] Sample mixtures were combined with 10 pl of reaction mixture containing: 1x Phi29 buffer, the 9 phosphorothioate modified primers at a concentration of 10 pM each, 0.4 mg / ml BSA, 2 mM of dNTPs and 1 U / pl of Phi29 DNA polymerase (New England Biolabs, St Quentin in Yvelines, France). The reactions were carried out at 30 ° C for 18 hours and terminated at 65 ° C for 10 minutes to inactivate the Phi29 DNA polymerase. RCA products are multiviters of HBV that can be digested by the restriction enzyme Spel. This restriction site being unique within the HBV genome, 3200 bp products corresponding to a complete HBV genome can be obtained.

Sequences and positions of primers used for RCA.

 Name

 Sequence (15 nt) Position in the HBV genome

 RCA1

 5'-AATCCTCACAATA * C * C-3 '226-240

 RCA2

 5'-GAT GGGAT GGGAA * T * A-3 '615-601

 RCA3

 5'-CCTATGGGAGTGG * G * C-3 '637-652

 RCA4

 5-'GCAACGGGGTAAA * G * G-3 '1154-1140

 RCA5

 5'-ATGCAACTTTTTC * A * C-3 '1814-1829

 RCA6

 5'-TCCAAATTCTTTA * T * A-3 '1916-1930

 RCA7

 5'-TAGAAGAAGAACT * C * C-3 '2374-2389

 RCA8

 5'-AGAATATGGTGAC * C * C-3 '2820-2834

 RCA9

 5'-TAAGAGACAGTCA * T * C-3 '3188-3202

PCR amplification of the complete HBV genome

[0113] The full length HBV genome was amplified by using a single stage PCR procedure using P1 P2 nar primers (5'-CCG GAA AGC TTA TGC TCT TCT TTT TCA CCT CTG CCC TAA TCA), P2 nar (5'-CCG GAG AGC TCG AGC TCT TCA AAA AGT TGG CAT GGT GCT GG-3 ') under the conditions described by Gunther et al (1995) and the FastStart high fidelity PCR system (Roche Diagnostics, Meylan, France ). The amplification products were developed on a 1% agarose gel and stained with ethidium bromide.

Analysis of PCR products and RCA products by Southern blotting

[0114] Five to ten microliters of the PCR products or 2 pl of RCA products were subjected to Southern electrophoresis and transfer using a specific S gene oligonucleotide probe labeled with 32P-dCTP by the terminal deoxynucleotide transferase (Roche Diagnostics, Boehringer Mannheim, Meylan, F).

Immunofluorescence or IHC in liver sections for surface antigen and HBV core antigen

[0115] Sections of frozen liver tissue 5 micrometers thick were fixed in acetone for 10 minutes at -20 ° C, blocked for 10 min with 3% PBS-BSA, and incubated for 45 min at room temperature with polyclonal rabbit antiserum antibodies raised against HBV nucleus antigen (1/2 dilution) (Serotec, Cergy St Christophe, France) or with mouse monoclonal antibodies raised against hepatitis Be surface antigen (dilution 1/40) (Dako, Trappes, F). After this incubation, the glass coverslips were washed in PBS and labeled with fluorescent conjugated goat anti-mouse or rabbit anti-rabbit antibody (1/100) (BIORAD, Marne La Coquette, France). The glass coverslips were tined with Evans blue, mounted and finally examined with a Leica DM RXE confocal microscope. For the liver tissues embedded in paraffin, the liver sections were dewaxed in xylene and rehydrated in graduated ethanol.

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Endogenous peroxidase activities were blocked with 0.3% hydrogen peroxide in methanol for 30 minutes at room temperature before labeling.

Observation by electron microscopy

[0116] Three milliliters of HBV and HBsAg positive DNA from cynomolgus macaque serum were centrifuged for 4 hours at 40,000 rpm, 4 ° C. The sediments were suspended in 500 ml of TNE (Tris pH 7.5 20 mM, 100 mM NaCl, 1 mM EDTA) and then diluted in 20 ml of TNE. A second centrifugation was performed at 40,000 rpm overnight at 4 ° C. The sediments containing the viral particles were resuspended in 300 ml of TNE and stored at -80 ° C.

[0117] The purification product was charged on a sucrose gradient from 10% to 60% and centrifuged at 100,000 g for 16 h. Twenty 0.6 ml fractions were collected and tested by PCR for HBV DNA with primers located in the HBV S and C gene. The positive fractions were loaded on carbon-coated gratings and stained with 4% phosphotungstic acid, pH 7.2 before examination by electron microscopy (Jeol 100 CX).

Preparation of cynomolgus macaque hepatocytes

[0118] Hepatocytes were isolated from M. cynomolgus after anesthesia and the liver was surgically removed. Cells were isolated by a two-stage collagenase perfusion procedure (Gibco, Cergy-Pontoise, France) as previously described (Guguen-Guillouzo, 1992). Freshly isolated hepatocytes were seeded in six-well plates at a density of 1.8x106 viable cells / cm2. This high density is essential for long-term survival of primary hepatocytes (Rumin et al., 1996). Adhesion was performed overnight in a Williams medium supplemented with glutamine (Biomedia, Boussens, F) supplemented with 100 Ul / ml penicillin and 100 mg / ml streptomycin (Gibco, Cergy Pontoise, F), 5 mg / l of bovine insulin (Sigma), 0.35% sodium bicarbonate and 10% heat-inactivated fetal calf serum (FCS) (Gibco Cergy-Pontoise, F). The following day, the medium was replaced by William E supplemented as described above, plus 2% dimethylsulfoxide (DMSO; Sigma, St Quentin, F), and 5x10-5 M hydrocortisone hemisuccinate (Upjohn, SERB, Paris, F). The cells were infected 1 day after planting as follows. The cultures were incubated for 20 hours at 37 ° C in the presence of 100 ml of the inoculum and 900 ml of fresh medium per well. Uninfected control cells were incubated in fresh medium plus 2% normal human serum. The supernatant from each culture was collected on day 1 and the cells were washed five times with phosphate buffered saline (PBS) and then kept in William's medium supplemented as before. Supernatants from each culture were then collected on days 3, 5, 7, 10 and 12, and analyzed for HBsAg using Monolisa HBsAg Plus (BIORAD, Marne la Coquette, France) and for vHb DNA by PCR.

"Pass" in vitro

[0119] A liver fragment of a cynomolgus macaque, positive for HBsAg and HBV DNA, was rinsed in PBS before inoculation and mixed using an Ultra-Turrax T25 homogenizer (Karayannis et al, 1989). The homogenate was clarified by centrifugation at 4 ° C at 6000 rpm for 30 min. The primary hepatocytes of M. cynomolgus were infected with 100 ml of liver extract in PBS (20%, w / v).

"Pass" in vivo

[0120] Two groups of M. sylvanus were used in this experiment (Atlas Mountains, Morocco). In order to study whether the HBV detected in M. cynomolgus was infectious to other macaques, 3 M. sylvanus were used which received a serum group of M. cynomolgus containing 103 particles / ml. In parallel, the same viral and biological parameters were monitored in a control group of 3 M. sylvanus that received no treatment. In vivo experiments were performed under general anesthesia using Imalgene (Merial, Lyon, France) containing ketamine (1 mg / kg).

Tracking infected animals

[0121] The infection was monitored by weekly blood tests of M. sylvanus (5 to 10 ml). The transaminases were controlled as well! as HBsAg and HBV DNA by quantification with Amplicor / monitor (Roche, Meylan, F) and qualitative PCR in the HBV S and C gene.

Sequencing

[0122] PCR products were sequenced by Genome Express (Meylan, F) using Biosystem 373A automatic sequencing. Sequence analyzes were performed using Mac Vector 6.5.1 and alignment was performed using the "BLAST" program in Genbank. .

Cloning

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[0123] PCR products (subgenomic or complete HBV genomes) were purified using "Qiaquick PCR purification kit" (Qiagen, Courtaboeuf, France). In all cases, 25 ng of plasmid DNA was ligated with the insert in a ratio in excess of 1 to 3 compared to the vector with T4 DNA ligase in a 50 mM Tris-HCl buffer pH 7.5, MgCl2 10 mM, 3% BSA containing 1 mM DTT, 0.1 mM ATP. Ligation was performed for 16 hours at 16 ° C.

Transformation

[0124] The plasmid containing the PCR product was introduced into chemocompetent bacteria with pCAP Le plasmide insert (from Escherichia coli M15) following the kit instructions (Promega, Lyon, France). Plasmid extraction was performed following the manufacturers instructions.

Amplicon transfection in Huh7 cells

[0125] The complete genome of HBV generated by PCR was purified using "Qiaquick PCR purification kit" (Qiagen, Courataboeuf, F) and digested for 20 hours with Narl (Promega, Lyon, France). Therefore, the DNA was diluted to a final concentration of 0.25 pg / pl. Huh7 cells were grown in a 25 cm2 flask containing 1300000 cells per flask. The transfection was done using Fugen following the Roche Applied protocol (Meylan, France) 24 hours after the start of the culture. HuH7 cells were collected and kept in a medium containing 500 ml of DMEM / HamF12, 50 ml of SVF, 10 ml of glutamine (200 mM), 5 ml of HEPES (1M), 2.5 ml of penicillin / streptomycin and 2.5 ml of sodium pyruvate. After 60 hours, the cells were trypsinized and resuspended in culture medium concentrated twice with DNase I and incubated for 45 minutes at 37 ° C. After granulation, the cells were resuspended in culture medium and distributed in 96 collagen coated wells. Therefore, cell cultures were treated for 4 days with various antiviral medications (lamivudine and adefovir). Four days after washing the treatment cells with PS and 50 pl of extraction buffer (25 mM Tris, 0.5 mM CaCl 2, 2.5 mM MgCh, pH 8, 0.4 mg / ml DNase, were added, 0.4 mg / ml RNase, 1% NP40). The cells were incubated for 1 hour at 37 ° C under stirring, then 15 pl of 0.4 M NaOH was added and the incubation lasted an additional 1 hour at 37 ° C. The alkalization was stopped with 15 pl of 1 M tris (pH 7). 5 pl of extracted DNA was used for quantification of HBV DNA by real-time PCR.

RESULTS

HBV isolation in cynomolgus macaques (Figure 2)

[0126] Biological samples from a total of 50 cynomolgus macaques were included in the study (liver and serum). Subgenomic PCR (see Tables 2 and 3 for primer sequences) and HBsAg detection using the Ortho Diagnostic test allowed to identify HBV markers among 42% (21/50) of the animals tested. Quantitative PCR by Amplicor / monitor (Roche) and Light Cycler PCR indicated viral HBV loads between macaques ranging from 102 to 104 copies of HBV aDn per ml of serum. Light Cycler PCR for the detection of HBV DNA of 10 ng of total liver DNA was estimated at 0.2 to 102 copies of HBV per hepatocyte.

Table 2: direct primers

Sequences 5'-3 '______________________________________________________

L1: 5 '- TCC TGC TGG TGG GCT CCA GTT CA - 3'

Pol1: 5 '- CCT GCT GGT GGC TCC AGT TC - 3'

Pol4: 5 '- CTC ACA ATA CCG CAG AGT CTA GAC T - 3'

Pol3: 5 '- CAA GGT ATG TTG CCC GTTT GTC L2: 5' - CCT GTA TTC CCA TCC CAT C - 3 '

Fw S: 5 '- GGA GTG GGC CTC AGC CCG TTT CTC -3'

E1: 5 '- TAA AAC AAT GCA TGA ACC TTT ACC CCG TTG C - 3'

Por5: 5 '- CAA GTG TTT GCT GAC GCA - 3'

R5: 5 '- AAG TGT TTG CTG ACG CAA CC - 3'

P197: 5 '- CCA TAC TGC GGA ACT CCT - 3'

B1: 5 '- GGC AGC ACA SCC TAG CAG CCA TGG - 3'

C1: 5 '- ACM TCS TTT CCA TGG CTG CTA GG - 3'

68: 5 '- CAT AAG AGG ACT CTT GGA TC - 3'

A3: 5 '- TGC GCA CCG CGG CCG CGC AAC TTT TTC ACT CTG CC - 3'

P'1: 5'-TTT TTC ACC TCT GCCTAATCAT- 3 '

P1: 5 '- CCC GAA AGC TTA TGC TCT TTT TCA CCT CTG CCT AAT CAT C - 3' C2: 5 '- CCT TCC GTC AGA GAT CTC C - 3'

Fw C: 5 '- TCG GAG TGT GGA TTC GCA CTC CTC - 3'

TP2: 5 '- ACC ACC AAA TGC CCC TAT CTT A - 3'

C3: 5'-CCT ATC TTA TCA ACA CTT CC - 3 '

genomic position

55-76

58-77

230-254

455-476

597-615

645-668

1134-1154

1178-1197

1179- 1200 1268-1287 1372-1386 1363-1386 1653-1672 1817-1843 1821-1841 1824-1843 1973-1992 2267-2290 2299-2320 2314-2333

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Four. Five

PS1: 5 '- GGG TCA CCA TAT TCTT GGG AA - 3'

PS4: 5'-GGA ACA AGA GCT ACA GCA TG - 3 '

TPR 1: 5'-TCG GGA AAG AAT CCC AGA GGA TTG G - 3 'Fw TPR 2: 5' - TGG GGT GGA GCC CTC AGG C - 3 '

2830-2850

2833-2852

2909-2923

3077-3095

Table 3: reverse primers

Sequences 5'-3 '______________________________________________________

R3: 5 '- GGC TCA GTT TAC TAG TGC CAT TTG T - 3'

Rev S: 5 '- GCC CCC AAT ACC ACA TCA TCC ATA - 3'

By 4: 5 '- TAC CCA AAG ACA AAA GAA AAT TGG - 3'

RV5: 5 '- AAG TGT TTG CTG ACG CAA CC - 3'

P198: 5 '- TTT TGC TCG CAG CCG GTC TG - 3'

P2: 5 '- CCG GAG AGC TCA TGC TCT TCA AAA AGT TGC ATG GTG CTG GTG - 3'

P201: 5 '- ATT AGG CAG AGG TGA AAA AG - 3'

P'2: 5'-ATG ATT AGG CAG AGG TGA AAA A - 3 '

67: 5 '- GTG GAG TTA CTC TCG TTT TTG CC - 3'

D2: 5 '- CTA AGG GTC GAC GAT ACA GAG GTC AGG CG - 3'

C6: 5'- AAG AAC TCC CTC GCC TCG - 3 '

Rev C: 5 '- GAT GAC TGA CTT CCT CCT CTG CGA GGA - 3'

C7: 5 '- GGG GCT TTA TTC CTC TAC AGT ACC T- 3'

Ps5: 5'-GGT TGA AGT CCC AAT CTG GAT - 3 '

Rev TPR 2: 5 'GCC TGA GGG CTC CAC CCC A - 3'

genomic position

693-669

763-739

828-805

1197-1178

1295-1394

1823-1803

1841-1822

1842-1821 1959-1937 2016-2000 2397-2380 2412-2388 2516-2492 2992-2972 3095-3077

[0127] To obtain the complete sequence of circulating HBV in cynomolgus macaques, the amplification of the complete HBV was performed using the procedure described by Gunther (1995) using primers P1 nar and P2 nar in liver DNA extracts from animals of 4 animals (38, 40, BL13 and BL14).

[0128] BL13 and BL14 were sylvanus macaques infected with the HBV of cynomolgus macaques. We were able to amplify 3200 bp amplicons, which hybridize with a HBV DNA probe after Southern blotting (Figure 3). Sequencing was performed directly on the amplicon without donation.

Sequence analysis

[0129] The amplified HBV sequence from 38 cynomolgus macaque liver tissue analyzed by BLAST allowed us to establish that this sequence is close to HBV D genotype. The total length is 3182 bp. A 33 bp deletion of the preS1 region of nucleotide 1905 and 1938 was observed. This deletion is observed among all non-human HBV genomes, but also in the HBV D genotype.

[0130] The nucleotide substitutions observed do not modify the open reading frames of HBV.

[0131] The analysis in the Genbank showed that HBV isolated from M. cynomolgus is very close to a genotype D HBV sequence that circulates between European IVDU (U 95551 / gi 2182117) (Figure 4). Minor substitutions were observed compared to the sequence already described. Arginine at position 122 and lysine at position 160 classified this HBV genome as subtype ayw3. Phylogenetic analysis of the sequences of the S gene confirmed the classification of protelna S among genotype D. The open reading frame of the viral polymerase encompasses the entire S gene, part of the X gene and the C-terminus of the nucleus gene ( 48aa). The encoded protein of the viral polymerase is identical to the HBV / ayw3 D genotype. A substitution of a guanine to adenine at position 1479 (within the proline X) and a substitution of proline to serine at position 67 (within the preS1 proline) were identified. The G1479A substitution does not lead to any loss of function or expression of the prothena X. The same HBV sequence was isolated with 100% homolog of BL14, a sylvanus macaque inoculated with a group of M cynomolgus serum, Island HBV positive Mauricio.

[0132] The complete genomes of HBV or subgenomic amplicons have been cloned and a number of sequences analyzed to confirm the results obtained. The newly generated sequences that cover 3/4 of the HBV genome were produced and coincided with the sequence obtained directly.

Quantification of HBV in both models

[0133] For cynomolgus macaques for which serum was available the Amplicor Monitor test (Roche) allowed to estimate the viral load in a range between 102 to 104 copies / ml.

[0134] The Light cycler PCR performed on liver extracts allowed to estimate the number of HBV copies in the Cercopitheque cephus over 1000 per mg of liver, which is a thousand times higher compared to the cynomolgus macaque. In the Cercopitheque model, CCCDNA could be detected by PCR (Werle et al, 2002). The transference

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of points made in liver extracts of both species was sensitive enough to estimate the amount of HBV DNA present in infected animals.

HBV protein expression in the liver of C. cephus and M cynomolgus

[0135] Histological study after staining with Hematoxylin / eosin showed severe hepatitis lesions in C cephus, while none of the chronically infected macaques showed liver lesions. Staining by IF or IHC revealed a strong expression of HBsAg in approximately 30% of hepatocytes (Figure 5). HBcAg was also detected in 10% of hepatocytes.

Infectivity of HBV isolated in M. cynomolgus

In vivo infection of macaque sylvanus by a group of cynomolgus macaques, positive in HBV DNA, positive in HBsAg

[0136] VHB virus particles were purified by ultracentrifugation (10X concentration) of cynomolgus macaque serum (HBsAg and HBV DNA positive). The concentrated material was then charged in a 10% to 60% sucrose gradient and centrifuged. Twenty 0.6 ml fractions were collected and then tested by PCR in the HBV S and C genes. Fractions 12 and 14 corresponding respectively to 42.5% and 48% sucrose were positive in PCR (Figure 6).

[0137] Positive fractions were observed by electron microscopy (Figure 7). In fractions 12 and 14, particles whose shape and size can correspond to Dane particles (Figure 7) could be observed. In fraction 4 (22.5% sucrose) spheres could be observed that may correspond to the 20 nm spheres described during HBV infections.

Macaque sylvanus infection by HBV particles of M cynomolgus.

[0138] Productive HBV infection was obtained in macaque sylvanus after infection with a group of cynomolgus macaques, as controlled by markers of HBV infection (Figures 8 and 9). Viral sequences were detected after infection for at least 9 weeks in 3/3 infected animals. The 1 ml inoculum contained a concentration of 103 copies per ml, 9 weeks after infection, it was estimated, for example, for the BL14 animal, that the viral load was 104 copies per ml. A peak of transaminases (approximately 250 U / L) was measured in the ninth week after infection in BL12 M. sylvanus and in the third week in BL13 and 14 of M. sylvanus. Histological examination at necropsy (32 weeks) showed lesions similar to those observed after acute hepatitis in humans (lymphocyte infiltrate, cleared hepatocytes, modification of parenchymal structure). No modification was observed in uninfected animals.

[0139] The detection of HBsAg that appears in the fourth to the ninth week after infection suggests a replication of active HBV after infection. HBsAg and HcAg were detected by immunofluorescence as illustrated in Figure 10.

In vitro infection of a primary culture of hepatocytes of M. cynomolgus with HBV isolated from cynomolgus macaque from the same isolate.

[0140] In order to study the infectious property of isolated HBV virus, primary hepatocytes of M. cynomolgus were incubated in culture with liver extract of an M. cynomolgus with positive HBV. Viral HBV sequences were detected in the cell supernatant after infection and intracellularly (Figure 11).

[0141] HBsAg was also monitored in the supernatant of infected primary hepatocytes after infection as illustrated (Figure 12). A second pass was made in primary culture of M cynomolgus hepatocytes successfully using HBVA positive DNA supernatant, HBsAg of infected cells.

Establishment of a macaque model of HBV infection

[0142] Two different approaches are possible for the development of a macaque model of HBV infection.

[0143] A first approach is based on the in vitro production of infectious particles. Isolates of Cercopithecidae with HBV described above, with a particle design as a 1.1 genome unit in a plasmid or a baculovirus, are used in parallel with a strain of human HBV (genotype D). These constructs are evaluated for their ability to replicate and infectivity in susceptible hepatoma cell lines (for example HepaRG) and in macaque hepatocytes (for example, primary macaque hepatocytes). The constructs that proved to be infectious are injected into macaques by serial intravenous injection of the HBV particles produced in vitro, thereby infecting the macaques.

[0144] A second approach is based on the use of infection particles for intrahepatic transfection, obtaining

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thus VHB infectious ape for the infection of macaque de novo.

[0145] In both approaches, the appearance of HBV markers for infection is monitored. The intrahepatic transfected macaques are monitored for two months with the clones of simian HBV isolates, as! As with human HBV. Serum samples are analyzed once a week in order to detect HBs and HBe antigens by ELISA and in order to determine the viral load by quantitative PCR. Biopsies are also performed in order to identify viral replicative intermediates, viral antigens and liver lesions. Serum samples at the peak of viremia are used to infect macaques, which are studied in a similar manner. Transfected and infected animals are also analyzed by serological infection markers after six months, and a histopathological examination of the liver is performed.

Follow-up of HBV infection of Sylvanus macaques after inoculation of the group of HBV particles of cynomolgus macaques or a group of HBV particles of humans

[0146] A group of 3 sylvanus macaques (BL 01, 03, 04) were inoculated intravenously with a group of highly viremic human sera (109 copies / ml) and the infection was monitored by measuring the titers of AgHBs and transaminases The HBV viruses of the group are HBV of genotype D and the sequencing of the HBV sequence demonstrates that the nucleotide at position 1479 is a guanine.

[0147] The results of these experiments are shown in Figure 13.

[0148] This figure illustrates for each of Sylvanus BL 01, BL 03 and BL 04 macaques the change in AgHB titles (left panel) and transaminase titres (right panel) during the days following HBV inoculation .

[0149] This figure shows a peak of HBsA just after the inoculation of HBV particles followed by a continuous decrease in HBV titers until HBsAgs can no longer be detected after approximately 3 weeks after infection. A peak of transaminases (reaching 292 U / L) was observed in the 3 animals after the disappearance of HBsAg.

[0150] A weak PCR signal for HBV DNA was detectable for 32 weeks (S and C genes) (data not shown).

[0151] This pattern is typical of a rinse of the originally inoculated HBV particles (sera of inoculated human HBV intravenously to sylvanus macaques lead to an inoculum disappearance profile without "de novo" replication) and suggests that HBV of Human sera cannot trigger a productive infection, that is, human sera viruses are unable to replicate efficiently.

[0152] These results confirm a study conducted by Lazizi et al. 1993, which also showed the lack of sensitivity to infection of a closely related macaque species (Macaca Mulata) by intravenous inoculation of the virus. In this study, HBsAg and HBeAg were detectable for 3 and 2 weeks, respectively, and viral sequences were detectable for 3 months, demonstrating a sequestration of viral particles after inoculation. At the time of sacrifice, 9 months after infection, the liver sections were negative for the staining of HBs and HBc.

[0153] A group of 3 macaques (BL 12, 13, 14) were inoculated intravenously with one ml of a group of Cynomolgus macaque sera naturally infected with HBV, said group of sera comprising 103 copies of HBV / ml . The genomic sequence of vHb particles from the sera group is shown in SEQ ID NO: 1 (the sera group comes from Cynomolgus Macaques that were previously identified as naturally infected by HBV particles that have a SEQ ID genome. NO: 1).

[0154] The results of these experiments are shown in Figure 14.

[0155] This figure illustrates for each of the Sylvanus BL 12, BL 13 and BL 14 macaques the change in the titles of AgHBs (left panel) and transaminases titles (right panel) in the days following the inoculation of HBV.

[0156] This figure shows a peak of HBsA just after the inoculation of HBV particles followed by a continuous decrease in HBV titres until HBs can no longer be detected after approximately 3 weeks after infection. A peak of transaminases (reaching 292 U / L) was observed in the 3 animals after the disappearance of HBsAg.

[0157] After inoculation with the cynomolgus macaque sera group comprising only 103 copies of HBV / ml, HBsAg was detected at individual time points (4 or 7 weeks after inoculation), which is indicative of production "de novo" of HBsAg after infection. A peak of transaminases (250 U / L) was observed after 9 weeks for BL12 and the third week for BL13 and BL14. Nine months after infection, the staining of HBsAg and HBcAg was positive in liver sections of animals.

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[0158] In addition, viral DNA sequences were detected by PCR that recognize HBV S and C genes throughout the follow-up. After 9 weeks, HBV DNA was quantified using a commercial assay (Amplicor Monitor) in M. sylvanus BL14 that gave a titer of 1x103 copies / ml. This title is not insignificant compared to the inoculum containing 103 copies, and a "de novo" production of new genomic sequences is confirmed.

[0159] In general, the results demonstrate that a group of sera comprising as few as 103 copies of HBV with a genomic sequence of SEQ ID NO: 1 is capable of initiating a productive infection in sylvanus macaques, while a group of sera Highly viremic humans comprising 109 copies of HBV of genotype D cannot trigger infection in sylvanus macaques. In addition, these results show that HBV with the genomic sequence of SEQ ID NO: 1 is particularly well adapted to infect macaques, particularly cynomolgus macaques and sylvanus macaques.

[0160] Direct inoculation of HBV DNA SEQ ID NO: 1 was also performed in the liver of sylvanus macaques and it was shown to induce an infection (ie, a typical infection profile with persistence of HBsAg was observed (even if sporadic ), increase in HBV DNA and persistence of markers at the time of slaughter (9 months after inoculation) as demonstrated by i) the presence of HBV DNA in the liver detected by PCR, and ii) HBsAg and HBcAg detectable by immunofluorescence in liver sections).

[0161] Together, these experiments provide evidence that human HBV or human HBV DNA particles are not infectious to macaques in the absence of adaptation mutations and that HBV DNA of SEQ ID NO: 1 is adjusted to infect macaques

REFERENCES

[0162]

- Barker, L. F., Chisari, F. V., McGrath, P. P., Dalgard, D. W., Kirschstein, R. L., Almeida, J. D., Edington, T. S., Sharp, D. G. and Peterson, M. R. (1973). "Transmission of type B viral hepatitis to chimpanzees". J Infect Dis. 127. (6). 64862.

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- Beasley, R. P. (1988). "Hepatitis B virus. The major etiology of hepatocellular carcinoma." Cancer. 61. (10). 1942-56.

- Bertoni R, Sette A, Sidney J, Guidotti LG, Shapiro M, Purcell R, Chisari FV. (1998). Human class I supertypes and CTL repertoires extend to chimpanzees. J Immunol. Oct 15; 161 (8): 4447-55.

- Blumberg et al .: A "new" antigen in leukemia sera, JAMA 191: 541, (1965)

- Chaoyin Cui et al., Journal of General Virology (2002), 83, 2773-2777, "The dominant hepatitis B virus genotype identified in Tibet is a C / D hybrid"

- Ganem, D. (1996) in Fields Virology, eds. Fields, B. N., Knipe, D. M. & Howley, P. M. (Lippincott-Raven, Philadelphia), pp. 2703-2737.

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- Gripon et al. (2002)

- Hu, X., Margolis, H. S., Purcell, R. H., Ebert, J. and Robertson, B. H. (2000). "Identification of hepatitis B virus indigenous to chimpanzees". Proc Natl Acad Sci U S A. 97. (4). 1661-4.

- Kock J, Nassal M, MacNelly S, Baumert TF, Blum HE, von Weizsacker F, Efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus, J Virol. 2001 Jun; 75 (11): 5084-9.

- Lanford, R. E., Chavez, D., Rico-Hesse, R. and Mootnick, A. (2000). "Hepadnavirus infection in captive gibbons". J Virol. 74. (6). 2955-9.

- Lemon, S. M. and Thomas, D. L. (1997). "Vaccines to prevent viral hepatitis". N Engl J Med. 336. (3). 196-204. MacDonald, D. M., Holmes, E. C., Lewis, J. C. and Simmonds, P. (2000). "Detection of hepatitis B virus infection in wild-born chimpanzees (Pan troglodytes verus): phylogenetic relationships with human and other primate genotypes". J Virol. 74. (9). 4253-7

- Maddrey, W. C. (2000). "Hepatitis B: an important public health issue". J Med Virol. 61. (3). 362-6.

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- Takahashi, K., Brotman, B., Usuda, S., Mishiro, S. and Prince, A. M. (2000). "Full-genome sequence analyzes of hepatitis B virus (HBV) strains recovered from chimpanzees infected in the wild: implications for an origin of HBV". Virology 267. (1). 58-64

- von Weizsacker et al., 2004

- Walter, E., Keist, R., Niederost, B., Pult, I. and Blum, H. E. (1996). "Hepatitis B virus infection of tupaia hepatocytes in vitro and in vivo". Hepatology 24. (1). 1-5.

- Warren, K. S., Heeney, J. L., Swan, R. A., Heriyanto and Verschoor, E. J. (1999). "A new group of hepadnaviruses naturally infecting orangutans (Pongo pygmaeus)". J Virol. 73. (9). 7860-5.

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Princess Takamatsu Symp. 12. 237-47.

- Wright et al., 1993

SEQUENCE LIST

[0163]

<110> INSTITUT NOTIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE <120> A new non-human primate model of HBV <130> BET11P0276 <160> 51

<170> PatentIn version 3.2 <210> 1 <211> 3182 <212> DNA

<213> hepatitis B virus <400> 1

Claims (19)

5 10 fifteen twenty 25 30 35 40 Four. Five 1. Use of a strain of HBV isolated from a human D genotype comprising the sequence SEQ ID NO: 1 to infect a macaque of the Cercopithecinae subfamily to produce a primate model for human HBV disease. 2. Use of an isolated macaque from the Cercopithecinae subfamily, preferably of the species Macaca sylvanus or Macaca cynomolgus, to generate a primate model for human HBV disease, in which said macaque is infected with a strain of HBV of a genotype D human comprising the sequence SEQ ID NO: 1. 3. Use, according to claim 1 or 2, said primate model having a viral load of HBV in the serum comprised between 102 and 108 genomic copies / ml. 4. Animal primate model for human HBV disease, wherein said primate is a macaque of the Cercopithecinae subfamily, preferably of the species Macaca sylvanus or Macaca cynomolgus, infected with a strain of HBV of a human D genotype comprising the sequence SEQ ID NO: 1. 5. Primate animal model according to claim 4, which is artificially infected with a strain of HBV isolated from a human D genotype, wherein said strain of HBV comprises the sequence SEQ ID NO: 1. 6. Primate animal model, according to claim 4 or 5, said primate model having a viral load of HBV in the serum comprised between 102 and 108 genomic copies / ml. 7. Procedure for providing a primate model for human HBV disease, comprising the following stages consisting of: - isolate a macaque from the Cercopithecinae subfamily; - infecting said primate with a strain of HBV isolated from a human D genotype comprising the sequence SEQ ID NO: 1; - test a viral load of at least 102 genomic copies of HBV / ml. 8. Method for evaluating a therapeutic agent, according to which, an animal model, according to any one of claims 4 to 6, is administered with defined doses, in a repeated dose or in repeated doses and at specific time intervals, of a therapeutic agent, Biological samples are taken before and at defined time intervals after the administration of said therapeutic agent and qualitative and quantitative measurements of the HBV viral proteins in said biological samples are made and compared. 9. Procedure to evaluate a vaccine to prevent a pathological reaction to a HBV infection, according to which: - a macaque is given with defined doses, in one dose or in repeated doses and at specific time intervals, of a vaccine, - said macaque is artificially infected with a strain of HBV isolated from a human D genotype comprising the sequence SEQ ID NO: 1, - biological samples are taken before and at defined time intervals after infection, and - qualitative and quantitative measurements of viral HBV proteins in these biological samples are carried out and compared. image 1 Polymerase: HBV polymerase: HBV DNA: RNA DNA: CORE RNA: NUCLEO pregenomic-RNA: pregenomic RNA Figure 1 image2 Macaque Macaque Liver nucleic acid extraction Immunohistochemistry HBcAg and HBsAg expression HBV complete genome amplification nfections Cloning Sequencing Subgenomic PCR Direct sequencing Tracking HBV infection markers image3 Figure 3 image4 image5 chimpance (AJ131587) 0.035 chimpance (DQG220) 0.032 genotype B (DQ0330) genotype B (D00331) genotype A (M57S83) 0.032 genotype A (X02763) genotype C (M12906) m.005 genotype C (X52939) Cynomolgus Macaque 0.037 0,) 07 genotype D (X02498) 0.01 s 0: 000 010 genotype D 0.009 ^ genotype genotype E (X75664) genotype 0.014 0.051 genotype 0.011 o.noa Woolly monkey 0: 194 Gibon 1 0.043 O.U 2 O.U Figure 4 image6 Figure 5 22.5% 42.5% 48% image7 Figure 6 image8 Weeks after liiii One thousand to infection Figure 9 BL12 CO image9 T TBs As Anti-I fee PCR ALT BL13 image10 0 2 4 6 Weeks after infection 8 ------------------------------ + 10 Figure 8 image11 image12 image13 image14 image15 image16 Serum transaminase activity BL13 image17 BL13 image18 image19